We use Drosophila as a model system to characterize the mechanisms underlying communication between cells, tissues, and organs.
Current Research Questions
1. What is the composition and organization of signaling networks?
2. What are the mechanisms by which cells compute incoming signals in time and space?
3. What are the signals that mediate communication between organs and how do they influence the development, regenerative properties, and physiology of individual organs?
4. How do environmental factors, such as nutrients and stress, influence homeostasis? Drosophila has an unrivalled arsenal of tools for both in vivo and in vitro functional studies.
Composition & Organization of Signaling Networks
To dissect signaling pathways, we use combinations of genome-wide RNAi and large-scale mass spectrometry to identify components of signaling networks, and characterize their activities using functional read-outs such as phosphorylation changes, transcriptional outputs, and cellular phenotypes. In particular, we are interested in identifying points of intersections, or "cross-talk nodes," between signaling pathways as cells within tissues are exposed to multiple signals that are somehow integrated into coherent responses. We apply these approaches to our ongoing Insulin/TOR pathway and Hippo network studies, as they play fundamental roles in coordinating growth and proliferation. To probe the functional redundancies within networks, we have begun using CRISPR-based knock out strategies in combination with RNAi to provide a powerful platform for synthetic functional screens. Finally, we are currently extending our signaling network studies beyond cell lines to complex tissues and are implementing a proximity labeling approach, based on the engineered peroxidase APEX, that allows characterization of subcellular proteomes in live tissues.
Cell Signaling in Time and Space
Understanding how intercellular signaling is able to produce defined cell fates or behavioral outcomes is a critical question in cell and developmental biology. Many years of studies have identified a small number of core signaling pathways: EGF, FGF, Cytokine JAK-STAT, JNK, Hedgehog, Hippo, Notch, NF-KB, Retinoic Acid, TGFβ and Wnt/Wingless; which are used in many different contexts to generate cell and tissue complexity. These studies have shown that 1. individual pathways can produce distinct responses and 2. that signaling crosstalk is key to generating diverse outcomes. It is now clear that the two work as an ensemble to generate the mature, functional organism. While a great deal is known about which signaling pathways are involved in which processes, it remains unclear how a combination of pathways function to affect cell fate and behavior. Manipulation of individual pathways over a timescale of hours or days gives an indication of how a specific pathway affects cell fate, and epistasis analyses can identify some relationships between pathways; however, primary signaling responses at the transcriptional level can occur on a timescale of minutes so secondary responses and sequential pathway crosstalk complicate interpretation of data from single, late time points. Dissecting the relationships between pathways and the flow of information that regulates cell fate requires a level of temporal and spatial resolution that can best be achieved by either time courses or ideally live imaging. We are developing a number of tools and approaches, based on tagging endogenous genes with dynamic fluorescence reporters that improve dramatically the temporal visualization of gene activities, to observe signaling at high spatiotemporal resolution in vivo. We are particularly interested in using these tools to analyze in the Drosophila gut how interactions between pathways are orchestrated to control stem cell proliferation during homeostasis and regeneration. In addition, to address questions about crosstalk and target co-regulation, we are mapping, using targeted DamID in gut stem cells, the targets of transcription factors regulated by the major proliferative signaling pathways.
Communication Between Organs
Organ-to-organ communications are critical to living systems and play major roles in homeostasis. For example, the vertebrate CNS receives information regarding thestatus of peripheral metabolic processes via hormonal signaling and direct macromolecular sensing. In addition, skeletal muscles produce various myokines that influence metabolic homeostasis, lifespan, and the progression of age-related diseases and aging in non-muscle tissues. Drosophila is a prime system for systematically identifying mechanisms involved in organ communication because libraries of transgenic RNAi lines are available that allow knockdown of any gene in an organ or tissue-specific manner. From such genetic screens we have already characterized a number of secreted factors (ImpL2/IGFBP; Myostatin/GDF11; Upd2/Leptin; Activin-beta) by which organs communicate their physiological state to others.These genetic screens are combined with RNAseq of specific organs to define the transcriptional signatures corresponding to their homeostatic states, mass spec analyses from blood to characterize secreted factors, and a novel proteomic method to discover organ communication factors. It is anticipated that these studies will reveal how subnetworks in one tissue influence subnetworks in a second tissue. Ultimately, this knowledge will generate testable hypotheses related to disease states (e.g., diabetes, aging, cancer), i.e., how biological processes observed in one tissue/organ (e.g., decreased cellular metabolism, mitochondrial dysfunction) may influence processes observed in a different tissue/organ.